弓形虫RH株微线体蛋白MIC3基因的克隆与表达

Cloning and Expression of MIC3 Gene of Toxoplasma gondii RH Strain

目的 克隆和表达弓形虫微线体蛋白MIC3基因。方法 从弓形虫RH株分离总的RNA,反转成cDNA.根据MIC3基因序列,设计合成一对引物,用聚合酶链式反应(PCR)方法从弓形虫cDNA中扩增MIC3基因片段,插入pGEM-T载体,并转化大肠杆菌Top10,经PCR、双酶切、测序验证后,将MIC3基因片段定向亚克隆到载体pET-28a中构建原核表达重组质粒pET-28a-MIC3,重组子在E.coli BL21中经IPTG诱导表达,并对表达产物进行SDS-PAGE和Western-blot分析。结果 从弓形虫RH株cDNA中扩增出792bp大小的MIC3基因片段并诱导表达27 300 Mr的重组MIC3蛋白。结论 成功构建和表达了弓形虫pET-28a-MIC3重组质粒,为弓形虫病诊断抗原和疫苗的研究莫定了基础。

Objective To clone and express the gene encoding micronemal protein MIC3 of Toxoplasma gondii RH.Methods Total RNA,isolated from T.gondii was reverse-transcribed into cDNAs.According to the sequence of MIC 3 gene,a pair of primers were designed and synthesized.A specific cDNA fragment of MIC3 gene was obtained by amplification of the cDNAs of T.gondii.After purification,the fragments of PCR products was cloned into pGEM-T vector,and then transferred into E.coli Top10.Positive clones was isolated and verified by PCR,restriction enzyme digestion and sequence analysis.The cDNA fragment was subcloned into pET-28a vector,and a recombinant pET-28a-MIC3 was constructed.The fusion protein was then expressed in E.coli BL21 and analyzed by means of SDS-PAGE.Results The 792bp cDNA fragment of MIC3 was amplified from the total mRNA of T.gondii RH,and the recombinant plasmid pET-28a-MIC3 was constructed.The expressed fusion protein had a molecular weight of 27 300 Mr.Conclusion Recombinant plasmid containing a gene fragment encoding MIC3 of T.gondii has been successfully constructed and expressed.This provides the basis for the diagnosis and production of vaccine against toxoplasynosis.